Airway hyperresponsiveness (AHR), a hallmark of asthma, consists of two features: heightened contraction of airway smooth muscle (ASM) upon exposure to non-specific stimuli, and ASM remodeling that reduces the effectiveness of bronchodilators. The cellular and molecular processes underlying these two undesirable changes in ASM, however, remain elusive. This project tests hypotheses that the augmented activation of the TMEM16A Ca2+-activated Cl- channel in asthma primes ASM to be hypercontractile, that TMEM16A functions as a common effector underlying neurotransmitter and inflammatory mediator-induced airway hypercontraction (accounting for the non-specific nature of AHR), and that it is a key molecule mediating ASM remodeling, contributing to persistent AHR. This proposal has 3 Aims. Aim 1: Because Tmem16a systemic null mice die shortly after their birth, studying the role of this gene in ASM in adulthood has not been possible. To circumvent this challenge, we have generated a mouse line whose TMEM16A in smooth muscle can be deleted specifically. This new unique mouse line will be used to establish that TMEM16A is required for Ca2+ spark (an endogenous localized Ca2+ signal for TMEM16A)-induced contraction in healthy adult mice, and that its upregulation causes ASM to be hypercontractile in asthma. The same hypothesis will also be tested using human ASM from healthy and asthmatic subjects. Aim 2: Our preliminary studies have shown that smooth muscle specific TMEM16A deletion or TMEM16A specific inhibitors largely inhibit mouse or human, respectively, airway contractions induced by neurotransmitter acetylcholine analog methacholine (Mch) or inflammatory mediators that activate G-protein couple receptors (GPCRs). We will use genetic, pharmacological and biophysics approaches to firmly establish that Mch and inflammatory mediators activate the GPCR-IP3 receptor-TMEM16A-L-type Ca2+ channel axis to contract airways, and that this axis is potentiated as a result of TMEM16A upregulation under asthma conditions. Aim 3: Since TMEM16A plays a pivotal role in cell proliferation, tumorgenesis, cancer progression and vascular remodeling, we propose that TMEM16A is required for ASM remodeling. To investigate this hypothesis, we will examine the effects of pharmacological inhibition of TMEM16A, or genetic deletion of TMEM16A in ASM cells on the remodeling. Since ASM remodeling persists in humans with severe asthma, we will study whether established ASM remodeling (in mice) can be reversed by either pharmacologically blocking TMEM16A or genetically deleting this channel in ASM cells via an inducible genetic knockout approach. We expect that our research will provide answers to two fundamental questions about asthma: why AHR is non-specific to different stimuli, and how ASM remodeling occurs. These answers should be instructive in the development of new anti-asthmatic strategies that not only have broad spectrum effectiveness, but also potentially reverse ASM remodeling, i.e. ?curable? asthma treatments.